cells in serum-free B27

Purpose To study the influence of serum-free B27 supplemented culture medium on corneal epithelial cells from limbal explants. Methods Human limbal tissues obtained from cadaveric donor eyes were used in this study. The morphological characteristics of cultivated epithelial cells were analyzed by phase contrast microscopy. Growth kinetics, bromodeoxyuridine (BrdU) labeling cell proliferation assay, and reverse transcriptase PCR (RT–PCR) for limbus and corneal markers were studied in serum-dependent and serum-free B27 supplemented corneal epithelial culture. The signaling pathway genes were analyzed by RT2 qPCR profiler array. Results The corneal epithelial cells morphology and mRNA expression of markers were similar in both the serum-dependent and serum-free B27 supplemented culture. The growth and proliferation of the serum-free B27 supplemented culture was significantly higher than that of the serum-dependent culture. The wnt, hedgehog, survival, NFkB, Jak-Stat, and calcium protein kinase C pathways were highly expressed in the serum-free B27 supplemented corneal epithelial culture. Conclusions Most signaling pathway genes are upfolded by B27 supplementation in the corneal epithelial cell culture; it could be an efficient replacement for serum.

Human limbal explant culture: The collected limbal tissue was washed thrice with Hanks balanced salt solution buffer (Sigma Chemicals). After careful removal of excessive sclera and conjunctiva, the tissue was cut into multiple bits using a sharp, sterile Bard-Parker blade (Niraj Industries, Faridabad, India). The tissue bits were placed on a culture plate (BD biosciences, San Jose, CA) using a sterile needle. The plate was incubated at 37 °C and 5% CO2 for 5 min for adhesion. The explants were covered with culture medium containing equal volumes of DMEM and F12 (Sigma Chemicals) containing 5 ng/ml of epidermal growth factor (EGF), 5 μg/ ml of insulin, 5 μg/ml of transferrin, 5 ng/ml of sodium selenite, 0.5 mg/ml of hydrocortisone, and 1% antibiotic solution (Sigma Chemicals). Ten percent FBS (Sigma Chemicals) was added to five cultures (serum-dependent culture; n=5) and 1% B27 supplement (Sigma Chemicals) was added to the other five cultures (serum-free B27 supplemented culture; n=5). The control samples were cultured without serum and/or any other supplement replacing serum (control culture; n=2). The plates were incubated at 37 °C and 5% CO2 with 95% humidity. The medium was changed once every two days and growth was monitored daily with an inverted phase contrast microscope (Nikon, Tokyo, Japan). Confluent cells were harvested for further molecular characterization. Growth kinetics: The outgrowth of all the cultures was photographed every second day; images were transferred to a computer and analyzed using quantity G area measurement software [5]. The mean radius of all the cultures was plotted against each day until they reached confluence. Cell proliferation assay: Cell proliferation was assessed by measuring 5-bromo-2-deoxyuridine (Qiagen, Santa Clara, CA) incorporation during DNA synthesis in proliferating cells. The detection of BrdU was performed according to the manufacturer's instruction and chased for 1-21 days. The BrdU labeling indices were assessed by counting the nuclei through a microscope using a 40× objective. The labeling index was expressed as the number of positively labeled nuclei/total number of nuclei×100%. RNA isolation: The cultures were trypsinised on the 8th day (limbal stem cells) and the 21st day (differentiated corneal cells) from both serum-dependent and serum-free B27 supplemented cultures. The RNA was isolated using the Rneasy (Qiagen) kit according to the manufacturer's instructions. For RT 2 qPCR array, the integrity and purity of the RNA were verified using a bioanalyzer chip (Agilent Technologies Genotypic, Bangalore, India). Reverse transcriptase PCR: The expression of marker genes (Bangalore Genei, Bangalore, India; Table 1) specific for limbal stem cells and corneal cells was studied by RT-PCR with the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control. Signal transduction pathway analysis: The RT 2 qPCR profiler Human Signal Transduction Pathway array (catalog number PAHS-014; SABiosciences, Frederick, MD), representing 84 genes involved in signal transduction pathways, plus five housekeeping genes and three controls, was used to analyze the effect of serum on signaling-related gene expression in human limbal and corneal epithelial cells. The total RNA was isolated from the limbus and corneal cells (serum-dependent and serum-free B27 supplemented culture) using the Rneasy Mini Kit (Qiagen). cDNA was generated from 1 µg total RNA using the RT 2 qPCR Array First Strand Kit in accordance with the manual. The template was combined with RT 2 SYBR Green/Fluorescein PCR master mix. Equal amounts of this mixture (25 μl) were added to each well of the RT 2 qPCR profiler plate containing the predispensed gene-specific primer sets, and the reaction was performed using a sequence detector (ABI 7500; Applied Biosystems, LabIndia, Chennai, India) according to the manufacturer's protocols. Data analysis was based on the ∆∆Ct method with the aid of an Excel (Microsoft Excel; Microsoft, Redmond, WA) spreadsheet containing algorithms provided by the manufacturer. The expression levels of the mRNA of each gene were normalized using the expression of the housekeeping gene GAPDH. A positive value indicates that the gene was upregulated and a negative value indicates that the gene was downregulated.
Statistical analysis: All experiments were performed in triplicate. The summary data were reported as the mean ±standard deviation (SD), and were compiled and analyzed on a computer (Microsoft Excel; Microsoft). The mean and SD were calculated for each group using the Student's t-test. Results were considered to be statistically significant when p<0.01. The results of RT 2 qPCR are indicated as "fold increase" (mRNA concentrations of serum-free B27 supplemented cultures divided by mRNA concentrations of serum-dependent controls).

RESULTS
Under microscopic observation, we noted epithelial migration from limbal explants at the end of 48 h in both serumdependent and serum-free B27 supplemented cultures ( Figure  1). By the end of the 15th day, 90%-100% confluent growth was seen. There was no growth in the control samples cultured without serum and/or any other supplement.
Growth kinetics: The cells cultured in serum-free B27 supplemented medium showed significantly higher growth after 12 days ( Figure 2). The growth rate was faster on cells cultured in a serum-free B27 supplemented culture when compared to a serum-dependent medium (p<0.005).

Comparison of signal transduction pathway genes
supporting the expansion of serum-dependent and serum-free B27 supplemented culture: The array experiment was performed in duplicate. A simple comparison was performed on data to assess the gene expression of a serum-free B27 supplemented culture in relation a serum-dependent culture as a control for limbal stem cells and differentiated corneal epithelial cells (Table 3). The differences in gene expression  between the serum-free B27 supplemented culture and the serum-dependent profile of limbal and corneal cells were studied (a more than twofold difference was considered significant). The raw data, i.e., the mean ∆∆Ct values of the genes, were normalized to the housekeeping gene GAPDH. All 84 genes were analyzed thoroughly based on their role in both the serum and serum-free conditions. Among these pathways, the most interesting and highly expressed were wnt, hedgehog, survival, NFkB, Jak-Stat, and the calcium protein kinase C pathways that have been discussed in this study ( Figure 5).

DISCUSSION
We have demonstrated the use of serum-free B27 supplemented medium for the growth of corneal epithelial cells. This serum-free medium supported the proliferation and viability of the cells. The cells expressed presumed limbal stem cell association markers and the cornea phenotype, suggesting that the serum-free B27 supplemented medium retained the stemness of cultured cells. The confluent culture was collected and RNA was isolated to analyze the signaling pathway genes involved in both serum-dependent and serumfree B27 supplemented cultures.
The signal transduction pathway genes involved in the growth of corneal epithelial cells help to determine their role in both serum-dependent and serum-free B27 supplemented corneal epithelial cultures. Among the 17 pathways, six pathways involved in the serum-free B27 supplemented culture were discussed, along with their roles in serum-free    In the serum-free condition of the corneal epithelial cells, the activation of wnt pathway plays a vital role by activating genes like Homo sapiens jun oncogene (JUN), which codes for a transcription factor called activator protein-1 (AP1) and helps in the differentiation, proliferation, and apoptosis of epithelial cells [6]. Corneal epithelial stem cell proliferation depends on the upregulation of paired box gene 6 (pax6) and downregulation of beta-catenin and lymphoid enhancerbinding factor 1 (Lef-1) [7]. The hedgehog pathway genes were 2 to 8 times upregulated in serum-free B27 supplemented limbal stem cells when compared with differentiated corneal epithelial cells of the same culture. Sonic hedgehog (Shh) is secreted by stem cells, inducing bone morphogenetic protein 4 (BMP4), and is involved in the self-renewal and development of the epithelium [8]. The wingless-type MMTV integration site family, member 1 (wnt1) and Wingless-type MMTV integration site family, member 21 (wnt2) genes of this pathway were found to play an equal role (12 times upregulated in relation to the serum-dependent culture) in the maintenance of stemness in limbal epithelial cells of the serum-free B27 supplemented culture. The cellular survival pathway consists of phosphoinositide 3-kinase/v-akt murine thymoma viral oncogene homolog 1 (PI3K/Akt), Janus kinase/ sarcoma proto oncogene (Jak/Src), and nuclear factor kappalight-chain-enhancer of activated B (NFkB) as three major groups of genes. The cyclin D1 (CCND1) gene is required for cell cycle G1/S transition [9]. Baculoviral Inhibitor of Apoptosis repeat proteins (Birc1) proteins contain BIR domains that can directly bind to active caspases and help in protein-protein interaction [10]. In the stem cell and progenitor cell compartments, the telomerase reverse transcriptase (TERT) gene prevents the adverse consequences of dysfunctional telomeres on cell viability and chromosomal stability [11], and enhances the cell cycle entry of quiescent epidermal stem cells [12]. The NFkB pathway genes in serumfree B27 supplemented cells had a distinct fold increase when compared with the control, and a few genes like interleukin 1 alpha (IL1A), interleukin 2 (IL2), lymphotoxin alpha (LTA), platelet/endothelial cell adhesion molecule 1 (PECAM1), and vascular cell adhesion molecule 1 (VCAM1) exhibited upfolded expression in both limbus and corneal cells. The inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase beta (IKBKB) gene produced an enzyme, IKK2inhibitor of nuclear factor kappa-B kinase subunit and activated a transcription factor called NFkB. Interleukin genes like IL1A, interleukin 8 (IL8), and tumor necrosis factor alpha (TNFα) present in the NFkB pathway encode for cytokines and chemokines involved in inflammatory processes [13,14]. They also help in the migration of progenitor and pluripotent stem cells [15]. The chemokine (C-X-C motif) ligand 9 (CXCL9) and interleukin 4 (IL4) genes of the Jak-Stat pathway played an important role in the development and organization of cells, which were upregulated by 12 times in serum-free B27 supplemented limbus culture [16]. Among the other five pathways, the calcium and protein kinase C pathway genes were highly expressed in serum free-B27 supplemented culture when compared to serum-dependent culture. The Homo sapiens V-fos FBJ murine osteosarcoma viral oncogene homolog (FOS) gene of the calcium and protein kinase C pathway belonged to the transcription factor family [17], which is highly upregulated in serum-free B27 supplemented limbal stem cell cultures.
In conclusion, the B27 supplement activated more signaling pathway genes, helping to provide a higher cell number, good capacity for proliferation, better quality, and more functional pieces of engineered corneal equivalents without the support of serum, a feeder layer, and/or BPE.